The present invention relates to a novel human Gp130 protein, DNA sequences coding for this protein, its use in therapy, particularly in in vitro fertilisation, as well as pharmaceutical formulations comprising such a protein.
Successful embryo implantation requires correct development of the pre-implantation embryo, resulting in a hatched blastocyst which is able to implant into receptive endometrium. A considerable body of data has been collected which supports the idea that soluble growth factors, if secreted by the uterine epithelium, act directly on the embryo to control this process (Anderson, E. D., J. Cellular Biochem., 53: 280-287 (1993) and Schultz, G. A. and Hevner, S., Mutat. Res., 296: 17-31 (1992))xe2x88x92.
In addition, developing embryos have been shown to produce a variety of cytokines which may act in an autocrine fashion on the endometrium to influence its receptivity. Examples of growth factors shown to be produced by human embryos include IL-1, IL-6, CSF-1 and TNF-xcex1 (Zolti et al, Fertil. Steril., 56 (1991) 265-272 and Witkin et al, J. Reprod. Immunol., 19 (1991) 85-93). TNF-xcex1 has been shown to be present in culture medium of human embryos up to the morula stage, but not that from the blastocyst (Lachappelle et al, Human Reproduction, 8: 1032-1038 (1993)). Production of cytokines by the embryo may therefore be regulated in a stage-specific manner.
Data on the possible direct effects of cytokines on embryos have come primarily from experiments in mice where many cytokines have beer shown to affect the development of preimplantation embryos in vitro. RFN-xcex3 and CSF-1, at physiological concentrations, inhibit the number of embryos developing to the blastocyst stage (Hill et al, J. Immunol., 139 (1987) 2250-2254). TNF-xcex1 has also been shown to have more subtle effects. Although TNF-xcex1 has no apparent effect on rates of blastocyst formation, it appears to specifically inhibit proliferation of cells contributing to the inner cell mass (ICM), which results in blastocysts with a reduced ICM (Pampfer et al, Endocrinology, 134: 206-212 (1994)).
Other growth factors also have specific effects on ICM cells. For instance, insulin-like growth factors 1 and 2 stimulate ICM proliferation, whereas leukaemia inhibitory factor (LIF) inhibit their differentiation (Harvey et al, Mol. Reprod. Dev., 31 (1992) 195-199).
As mentioned above, IL-6 is one of the growth factors which has been shown to be produced by human embryos. IL-6 is a protein which controls the proliferation and differentiation of many cell types in mammals, and in addition has a role in the control of the immune system. Binding of IL-6 to IL-6R initiates the association of IL-6R with a third component known as gp130, which actually transmits the signal through the cell membrane (Taga et al, PNAS, 89: 10998-11001). gp130 is a transmembrane protein, i.e. it extends through the membrane and projects into the cytoplasm, thus it has distinct domains. In this way, IL-6 #xe2x80x9csignalxe2x80x9d transmission is mediated by means of this protein.
EP-A-0411946 discloses a recombinant gp130 protein, as well as DNA sequences coding for such a protein and methods for its cloning.
Yasukawa et al, Immunology Letters, 31 (1992) 123-130, disclose a soluble, recombinant form of gp130, produced by removing the transmembrane and cytoplasmic regions of the membrane bound form of the protein.
Narazaki et al, Blood., 82, No 4 (1993) 1120-1126, disclose that soluble forms of gp130 exist and may have potential to inhibit signals normally mediated by transmembrane gp130.
The present invention relates to a novel human gp130 protein, DNA sequences coding for the protein, use of the protein in therapy, particularly in in vitro fertilization, and pharmaceutical compositions comprising the protein.
The invention further relates to the use of gp130 to antagonize the action of one or more growth factors, wherein the growth factors are IL-6, LIF, CNTF, Oncostatin M and IL-II.
The invention also relates to the use of gp130 to ensure the correct development of pre-implantation embryos.
The invention further relates to pharmaceutical compositions comprising gp130 together with pharmaceutically acceptable excipients.
The invention also relates to a method for antagonizing the action of one or more growth factors in a pre-implantation embryo which comprises the step of administering to the embryo gp130.
There has now been found a novel form of gp130, lacking the transmembrane portion, which form arises by means of alternative splicing of the sequence coding for the transmembrane gp130. This form of gp130 may bind to complexes of, for example, IL-6 receptor, resulting in a blocking of the association of the complex with transmembrane gp130.
The novel splicing pattern, as well as resulting in loss of the transmembrane domain, also results in a frameshift, leading to 45 new amino acids before an in frame stop codon.
Thus, the present invention provides gp130 which includes the following C-terminal sequence: (SEQ ID NO:1)
Glu Leu Lys Asn Thr Ser Gly Leu Met Phe Gln Ile Leu Gln Arg Val Ile Leu Pro Ser Gly His Leu Thr Leu Leu Gln Gly Thr Ile Leu Ile Gln Lys Ile Lys Cys Ile Gln Met Ala Ile Ser Leu Met
or a C-terminal sequence which is substantially homologous thereto.
Preferably, the novel gp130 of he invention has the above-noted sequence from amino ac position 614. In one embodiment the novel gp130 of the invention has a sequence at positions 1-613 substantially homologous to that shown in FIGS. 2A-2F (SEQ ID NO:6)
At the amino acid level, a protein sequence may be regarded as substantially homologous to another protein sequence if a significant number of the constituent amino acids exhibit homology. At least 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids may be homologous.
Thus, activation of the alternative splicing mechanism can result in the production of a novel gp130 in human blastocysts, resulting in inactivation of the effects of, for example, LIF, which in turn ma allow differentiation of the inner cell mass, allowing ICM differentiation to proceed. Clearly, therefore, the novel gp130 of the invention can be used in the treatment of preimplantation embryos to ensure correct differentiation and development prior to implantation in a subject.
In addition, the invention also provides a DNA sequence coding for a protein of the invention which sequence includes a sequence substantially homologous to: (SEQ ID NO:2)
GAA TTA AAA AAC ACA TCT GGC TA ATG TTC CAG ATC CTT CAA AGA GTC ATA TTG CCC AGT T CAC CTC ACA CTC CTC CAA GGC ACA ATT TTA ATT CAA G ATC AAA TGT ATT CAG ATG GCA ATT TCA CTG ATG TAA
xe2x80x9cDNA sequence substantially the samexe2x80x9d includes all other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequence or which are substantially homologous to such a sequence.
Sequences having substantial homology may be regarded as those which will hybridise to the nucleic acid sequence shown in FIGS. 2A-2F (SEQ ID NO:5) under stringent conditions (for example, at 35 to 65xc2x0 C. in a salt solution of about 0.9 M).
DNA constructs comprising DNA sequences of the invention form another aspect of the preset invention.
As discussed herein, the protein of the invention is useful in antagonising the action of certain growth factors, thus enabling certain development processes to be xe2x80x9cswitched onxe2x80x9d in preimplantation embryos. Thus, in a further aspect, the present invention provides the use of the protein of the invention in antagonising the action of one or more growth factors, including IL-6, Leukaemia Inhibitory Factor (LIF), Oncostatin Myciliary Neurotrophic Factor (CNTF) and IL-II.
In addition, the invention also provides the use of the protein of the invention in he manufacture of a medicament for use in ensuring correct development in preimplantation embryos. Preferably, the medicament is used to ensure that differentiation of the ICM commences at the correct time.
The medicament is preferably presented in the form of a pharmaceutical formulation comprising the protein of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical formulations form a yet further aspect of the present invention.
A final aspect of the present invention provides a method for antagonising the action of one or more growth factors which comprises the step of treating a pre-implantation embryo with the protein of the present invention, preferably in the form of a pharmaceutical formulation. Preferably the invention provides a method for ensuring correct development of a preimplantation embryo.
The invention will now be described by means of the following examples, which examples should not be construed as in any way limiting the present invention. The examples refer to the following figures which show: